Labs 10 and 11: Bacterial Transformation and DNA Purification

Transcription

1 Biology 107 General Biology Labs 10 and 11: Bacterial Transformation and DNA Purification Molecular biology often involves altering the genetic makeup of an organism in a directed way. In this lab exercise, we will introduce foreign DNA into a bacterial culture, select cells that take up our plasmid, and then use these cells to manufacture large quantities of plasmid DNA. These manipulations will enhance your understanding of molecular methods we discussed in lecture. Objectives When you have successfully completed this lab, you should be able to: 1. Introduce plasmid DNA into E. coli, and select for transformants. 2. Calculate a transformation efficiency for your experiment. 3. Explain the importance of the plasmid origin of replication and the selectable marker. 4. Amplify and purify your plasmid DNA from a single transformed colony. 5. Explain the chemistry involved in each step of your plasmid purification. 6. Separate DNA molecules on an agarose gel. IMPORTANT: A portion of your grade for this laboratory depends on your ability to determine the number of cells in your original tube of bacterial culture, calculate a transformation efficiency, and recover your plasmid in the second week of the lab exercise. You are required to come to lab each week with a written protocol. Lab manuals may not be used during these exercises. Once each week, you will be required to visit the lab outside of your normally scheduled class time. If your experiments do not work correctly, you should be prepared to repeat them. Introduction As we have discussed in the lecture part of Biology 107, transformation is the process by which bacterial cells take up unpackaged (e.g., plasmid) DNA. When this DNA enters the cell, the bacteria gain the ability to express any genes that are encoded by the plasmid, producing proteins according to the recipes in the plasmid DNA. This allows us to determine which cells have taken up the plasmid - if the plasmid contains a recipe (gene) for a protein that detoxifies the antibiotic ampicillin, then cells that take up the plasmid will be able to survive in the presence of ampicillin (Figure 1). This gene is called a selectable marker because it allows the biologist to select cells that contain the plasmid. In addition to the selectable marker, plasmids contain DNA sequences called replication origins. These sequences are signals for the bacterial DNA replication proteins to begin duplicating the plasmid. The plasmids we are using have origins that direct the cells to maintain between ten and one hundred plasmid molecules per cell. This high copy number will be useful when we use the bacteria to amplify our plasmid. In the first week of this exercise, you will mix plasmid DNA with competent E. coli cells, and determine which cells take up the plasmid. By comparing the number of cells you started with and the number that took up the plasmid, you can calculate the transformation efficiency. 2004, University of Evansville Biology Department 58

2 59 many bacteria were in your starting solution. You also need to transform a separate sample of cells. Section A, Counting Bacteria, requires that you make serial dilutions and Section B, Transformation, requires you to mix cells and DNA and incubate them on ice for thirty minutes. To efficiently use our two-hour lab period, you should begin this incubation (outlined in Section B, below) first. All of the experiments in this exercise require students to use sterile technique. Sterile is defined as free from any living form of microbial life. Sterile or aseptic technique in the Biology 107 lab can be thought of as precautionary measures used to prevent contamination of the E. coli cultures used for transformation and counting. Figure 1. Transformation and selection. Remember, the micropipettor (Figure 2) you will use is a mechanical device and it is NOT sterile. The small plastic tips which you will place onto the end of The E. coli you will be using are treated to increase their efficiency of taking up foreign DNA. Untreated E. coli cells mixed with foreign DNA will provide transformed cells, but at a very low frequency. However, E. coli can be made competent to take up DNA by treating the cells with an agent such as CaCl 2 on ice. The Ca ++ ions form an ionic bridge between the negatively charged phosphate groups on the DNA and the membrane phospholipids. The cold temperature crystallizes the membrane and stabilizes this interaction. Subsequent heat shock of the cells causes some of the exposed cells to take up DNA. In the second week, you will use one of your transformed bacterial colonies to inoculate a culture. The bacteria you grow should contain your plasmid. To demonstrate this, you will purify your plasmid away from all of the other molecules in the cell and visualize it using gel electrophoresis. Procedures During the first lab period, you will be doing two things at the same time. You need to serially dilute untransformed bacteria so that you will know how Figure 2. Micropipettor with tip installed.

3 60 the pipettor ARE sterile. This means that when you slowly insert the sterile tip into the growth medium to remove 0.99 ml of sterile broth and set up your dilutions, only the tip can touch the liquid or any part of the inside of the flask. If you accidentally clunk any part of the micropipettor onto a sterile surface, such as the inside of the flask containing the sterile broth, you risk contaminating the broth for everyone else in your lab. If you make this mistake, tell your instructor. To minimize this potential problem, one member of each group should carefully watch the persons using the micropipettor and visually help guide them as they enter the flask. During this lab exercise, you will not refer to your lab manual. Before class each week, each student will individually prepare a protocol for the lab exercise. This protocol is a typed document that lists the steps of your experiment, and explains why each step is important. Your protocol does not have to be a long document, but it does have to be complete. Think back to the experiment in section B of the enzyme lab (page 39). A student protocol for this exercise might read as follows. 1. We will be carrying out four reactions, each with its own blank, so we will need eight test tubes for each group. Blank tubes will contain potato extract, but no substrate (catechol) so that the color due to the extract can be subtracted from the total color in the reaction tubes to give the color due to product formation. Mark the tubes as indicated in the data table. 2. Using glass pipettes to measure liquid volumes, make blank tubes containing water and potato extract according to the data table. Seal with parafilm and mix the blank tubes so that they are the same color throughout. 3. Set the spectrophotometer to 540 nm. This wavelength of light is absorbed by the product. The more absorbance we read, the more product is in the tube. 4. Zero the spectrophotometer with Blank 1. Any additional absorbance in tube 1 is due to product formation. 5. Begin making tube 1 by adding water and substrate to the tube. Do not add the extract (contains catecholase) until everything is ready. 6. Start the reaction by adding potato extract, sealing, and mixing the tube. Note the time the reaction began. Allow the reaction to continue converting substrate into product for three minutes. 7. As the three-minute time approaches, mix the tube again, and read the absorbance at exactly three minutes. Record the absorbance and time in the data table. The absorbance is in indication of how much product was formed. 8. Repeat steps 4 through 7 using tubes with different amounts of potato extract. Be sure to use the correct blank with each reaction tube. Adding more enzyme should increase the amount of product formed. This protocol, along with the data table, would allow you to do the experiment correctly. Writing it out beforehand would help you think about what you were going to do and why. Your protocol is not complete without an explanation of your experiment. You will need two protocols for week one - Counting Bacteria (including a dilution diagram), and Transformation. For the second lab week, you will need a data table for transformation results as well as Plasmid Purification and Gel Electrophoresis protocols. Remember, you will be using your written protocols, not your lab manual, so do a good job! Section A - Counting Bacteria How many bacteria are in a given sample? This question arises in the clinical microbiology laboratory, in public health debates, and in many molecular biology experiments. How might you answer this question? The most convenient method is to spread the cells on an agar plate and then incubate the plate overnight. Wherever a cell lands on the plate, a bacterial colony is present the next morning. For an example of plating cells, see page 378 in your textbook. Competent cells have been prepared by your instructor, and are stored on ice in the tube labeled Cells. The cells must be kept ice cold at all times.

4 If they are allowed to warm even slightly, your experiment will not work. Since your tube contains hundreds of millions of cells, counting each and every one is not really possible. Instead, we will dilute the cells a known amount, count the cells in the diluted sample, and calculate the number of cells in the original tube. The dilution calculations in the following example should be carefully reviewed before lab. If you want additional examples of dilution calculations, review your work from the first lab exercise. DILUTION SAMPLE CALCULATION Assume that a tube has 1 ml of medium containing 10 8 cells per ml. If you spread all these cells on a plate, they would be impossible to count. Since we can only really count about 100 (10 2 ) cells on each plate, we must dilute the cells in the original tube one million (10 6 ) fold (10 8 cells in the original tube divided by 10 6 = 10 2 cells on the plate) so that we can count them. Plating and counting the cells in a 10 6 fold dilution of our original cells will allow us to multiply by one million to get back to the number of cells per ml we started with. (100 cells on the plate multiplied by 10 6 = 10 8 cells in the original tube) A one-million-fold dilution is impossible to carry out in a single step without using large quantities of liquid. For example, diluting your original 0.1 ml one million fold in one step would require 99,999.9 ml or approximately 100 liters of medium. Instead, we will use serial dilutions. Before beginning your dilution, you should watch the instructor demonstrate the use of the micropipettors. Using these devices is an important skill in the biology laboratory, and your grade depends on careful measurement. At the worktable, select three sterile microtubes for each group and label them 10-2, 10-4, and Working sterilely, use the micropipettor to place 0.99 ml (990 µl) of sterile medium into each of the three sterile tubes. Remember that any bacteria introduced from your hands or elsewhere will throw off your count. Then, place 10 µl of your bacterial sample into your tube marked Note that this is a 100 (10 2 ) fold dilution of your original tube. Mix this tube well. Then, using a new sterile tip (why?) take 10 µl from this tube and place it in the tube marked 10-4, for a 10 4 dilution. Mix, and place 10 µl from this tube into the tube marked 10-6 for a 10 6 dilution. Now, still working sterilely, spread 200 µl of your 10-6 dilution onto an agar plate using the hockeystick method demonstrated by your instructor. Since you want to assure that you can count the cells on the plate, make another counting plate with 20 µl of your 10-6 dilution. For your protocol, make a diagram explaining your dilutions. Draw the tubes and plates you will use, the amounts of liquid in each transfer, and how many cells you expect in each tube or on each plate if your original solution has 10 9 cells per ml. Make sure the plates are properly labeled, and give them to your instructor for overnight incubation. Arrange for your lab group to visit the lab tomorrow to count the bacterial colonies on the plate, and if necessary to redo the experiment. Before you leave class, use your dilution diagram to think about how you will answer the following question: what was the number of cells per ml in the original tube? For example, if you count 17 cells in 100 μl of your 10-6 dilution, how many cells are in each ml of this dilution? How many cells are in each ml of the 10-4 dilution? How many cells per ml in the original tube? IN YOUR LAB REPORT, STATE THE NUMBER OF CELLS PER ml IN THE ORIGINAL TUBE. Section B - Transformation 61 DNA has been prepared for you by your instructor and 1 µl of DNA solution will be placed into a sterile 1.5 ml tube for each group. (One µl is ml and pronounced microliter. It is also referred to as λ or lambda.) Label the tube with your group number and get ready to place 0.1 ml (100 µl) of cells from the ice water bath into the tube. It is essential that the cells stay ice cold and sterile at all times. Quickly remove the tube labeled Cells from the ice bath, invert gently to mix, carefully open the cap and, using

5 the micropipettor, remove exactly 0.1 ml (100 µl) from the tube and place it in your labeled tube containing DNA. Gently mix the contents of the tube by flicking the side with your fingernail. Do not shake the tube or you will damage the bacterial cells. Immediately place the tube back into the ice bath and incubate it there for thirty minutes. This incubation allows the DNA to bind to the outside of the cell, and would be a good time to start your dilution experiment. After thirty minutes, heat shock the cells at exactly 42 C for exactly ninety seconds, then return to ice for two minutes. This causes the cells to take up the plasmid DNA. Then, add 0.9 ml of medium, and incubate at 37 C for a thirty to forty-five minute outgrowth. During the outgrowth, cells have an opportunity to express genes on the plasmid, making the protein responsible for conferring ampicillin resistance. Plate 0.1 ml of your transformation mix ON A PLATE CONTAINING AMPICILLIN using the same method you used in your dilution. Plate 0.01 ml on another plate, in case your first plate has too many colonies to count. Count the transformed colonies tomorrow. IN YOUR LAB REPORT, STATE: 1) THE NUMBER OF CELLS IN YOUR TRANSFORMATION TUBE; 2) THE NUMBER THAT TOOK UP THE PLASMID; AND 3) THE TRANSFOR- MATION EFFICIENCY (EXPRESSED AS A DECIMAL). Section C - Counting Colonies When you return to class to count your cells, remember that wherever a single cell landed on the surface of the plate, a colony of cells will be present the next day. When you count these colonies, you are counting all of the cells that were able to live on the plate where they were spread. You are estimating two quantities: the number of cells in the Cells tube, and the number of cells that took up the plasmid. For each of these estimates, you have two plates. You should see a 10-fold difference between similar plates. Example: If you count 37 colonies on the ampicillin plate which received 10 µl of transformed cells, you would expect about 370 colonies on the plate which received 100 µl. If, however, you counted 11 colonies on the plate which received 100 µl of transformed cells, you may find no colonies on the plate with the smaller volume. You expect a 10-fold difference, but you wonʼt always find it. In this lab, you should count all of the colonies that are possible to count Enter the number of colonies you count in the space below. If there are so many colonies on a plate that they are too close together to distinguish, make a note of that on your data sheet and use the count from the companion plate for your calculations. After you count the colonies on your plates, be sure to WASH YOUR HANDS WITH SOAP before leaving lab. Save your plates for next weekʼs lab exercise (See Section D). Plate Cells, 10 6 dilution 200µl Cells, 10 6 dilution 20µl Transformation 100µl Transformation 10µl Number of Colonies In order to know how well your experiment worked, you will need to compare your results to the results of others. Design a table to collect the transformation and cell count results from all of the groups in your lab section. Bring it to class next week. Section D - Plasmid Purification 62 When biologists introduce a plasmid into E. coli, it is usually because they want to use the bacterium as a means of storing and amplifying the plasmid. If the plasmid carries a DNA insert, then the insert is amplified along with the rest of the plasmid. This is a good way to make a large number of copies of an insert for further analysis. It is only useful, though, if there is a

6 63 convenient way to recover the plasmid from the bacteria. Remember, in the transformation experiment, you introduced plasmid molecules into bacterial cells. Each cell that took up a plasmid became a colony containing millions of cells after overnight growth. Since each of these cells has between ten and one hundred plasmid molecules, large amounts of plasmid (and insert) can be prepared quickly. During the second week of this lab exercise you will grow bacterial cells containing your plasmid, purify the plasmid DNA and visualize it using an agarose gel. Plasmid purification involves a number of steps, as described below, but the principle is very simple. Cells are collected, broken open, and plasmid DNA is separated from all of the other molecules in the cell. This procedure is outlined in Figure 3. Figure 4 on the following page shows an illustrated version of the steps outlined in Figure 3. The day before your lab period, someone from your group should come to the laboratory at the scheduled time. Make sure you have prepared for this meeting - you should know all the details about what you are doing, and why. Your instructor will have prepared a number of culture tubes containing sterile liquid medium that contains ampicillin. Working sterilely, inoculate a culture tube with a single bacterial colony. Simply touch the inoculating loop first to the colony then to the liquid in the tube. It is not necessary to move the whole colony, or even a visible clump of cells. Place the tube in the shaking water bath as directed, and allow to shake overnight at 37 C. At the beginning of your lab period, each group should take one 1.5 ml microtube, label it with their group number, and use a pipette to transfer 1.5 ml of the bacterial culture into the tube. Then, place the tube in the microcentrifuge, and your instructor will spin the tubes so that the cells form a pellet on the bottom of the tube. Each group should then recover their tube, pour off the medium without disturbing the pellet, and add another 1.5 ml of the same bacterial culture to the tube containing the pellet. After another centrifugation and complete removal of the medium (also called the supernatant) the tube will contain only the cells from 3 ml of bacterial culture. Next, we will add 250 µl of cell resuspension solution to the pellet. Use the vortex machine to get all of the cells back into suspension. When you are ready, add 250 µl of cell lysis solution and mix by inverting the tube four to five times. This solution has a very high ph and contains sodium dodecyl sulfate, a strongly amphipathic detergent that dissolved the cell membrane, releasing the contents. When you add this reagent, you should see the solution clear somewhat and become very viscous. The viscosity is caused by the bacterial chromosome, which we will precipitate and discard shortly. Treat the contents of the tube gently after lysis - DO NOT VORTEX. After your lysate has cleared, add 10 µl of alkaline protease solution to digest cellular protein, and incubate for five minutes at room temperature. Figure 3. Outline of Plasmid Purification. Add 350µl of neutralization solution to the lysate, and mix gently (do not vortex). This lowers the ph and most lipids precipitate (come out of solution).

7 64 Since the bacterial chromosome is attached to the membrane, it also precipitates in this step. Your instructor will centrifuge the tube for ten minutes, and this will again result in a pellet and a supernatant. This time it is the supernatant that contains the plasmid DNA. Move to the vacuum manifold (Figure 4 on the next page), obtain and label a minicolumn with your group number, install the minicolumn onto the vacuum manifold as directed, and carefully pour your cleared lysate into the column. The column contains resin that binds plasmid DNA. You will attach your plasmid DNA to the column and then wash away other impurities. Apply vacuum using the stopcock on the vacuum stem so that your lysate is drawn through the column. When all the liquid has been drawn through, release the vacuum. Then add 750 µl of column wash solution, apply vacuum to draw it over the column, and release the vacuum. Repeat the wash step, using only 250 µl of column wash solution for the second wash. When all of the wash solution has been removed from the column, release the vacuum, and transfer the column to a spin collection tube. Your instructor will spin the column briefly in the microcentrifuge to remove any remaining liquid. Then, transfer the column to a new 1.5 ml microtube (labeled with your group number), add 100 µl of water to the column, and allow your instructor to centrifuge the water through the column. Since the DNA does not bind to the column resin in the presence of pure water, it is released, and travels with the water into the centrifuge tube. You may now discard the column. Figure 4. Wizard SV Illustrated Protocol. Reproduced with permission from Promega Notes 59:10 (1996).

8 65 Section E - Gel Electrophoresis CAUTION: UV light and ethidium bromide are both hazardous. Listen carefully when your instructor explains how to minimize your exposure. A glance at the tube with your plasmid should convince you that a small volume of water containing plasmid DNA is not easy to distinguish from any other small volume of water. We need a way to visualize the DNA. To determine whether you have correctly purified your plasmid, combine 10 µl of your plasmid solution with 5 µl of blue loading buffer in a new 500µl microtube. Be careful, this buffer will stain your skin and clothing. Mix gently, centrifuge for 5 seconds, and load all 15 µl into a well of the agarose gel your instructor has prepared. Once all groups have loaded their DNA on the gel, an electric field will be applied, and negatively charged DNA will move toward the positive electrode. The gel matrix retards this movement, with larger DNA molecules retarded more than small molecules. The result is separation of DNA molecules by size (Figure 5). Your instructor will include on the gel a sample of the correct plasmid, and yours should run at the same size. DNA will be visualized using a UV transilluminator. The gel matrix prepared by your instructor contained a compound called ethidium bromide. This molecule binds strongly to DNA, and the bound complex fluoresces orange in the presence of UV light. We will place the gel on a UV light source, and any DNA in the gel will begin to glow brightly. Your instructor will post a photo of your gel on the course web site for you to include in your lab report. Figure 5. DNA separated by size on an agarose gel. This gel contains 20 lanes. Each sample is loaded into a well near the top of the gel. An electric field is applied, and the molecules move downward in each lane, with small molecules moving fastest. DNA is visualized under UV light in the presence of ethidium bromide. The far right and left lanes contain standards of known size. Lab Assignment This assignment is to be handed in the week of November 29, at the beginning of lab (before your quiz). Each student must complete this assignment individually. Write a brief introduction and a methods section explaining what you did in this experiment. Make sure these sections cover transformation, selection, plasmid purification and gel electrophoresis. Then, write a results section in which you report the number of cells per ml in the original tube of competent cells (the tube labeled Cells ). Use this number to determine how many cells you used for the transformation. Calculate how many of these cells were transformed. Why must you use data from other lab groups in your section to get an idea of how well your experiment worked? Your lab report will not be complete without an analysis of your data compared to class results. Return to section D of the enzyme lab exercise (pages 45-46). How can you use the class mean and standard deviation to tell whether your results make sense? Include in your results section a printed photo of your agarose gel showing that you recovered the same plasmid you received from the instructor in the first lab period. All text material for this assignment must be submitted to